Summary: Oxidation Resistant apoA1 Gene Delivery Stents Stent angioplasty has led to dramatic improvements in outcomes for coronary artery disease. Drug eluting stents (DES) have sharply reduced the incidence of in-stent restenosis (ISR). Nevertheless, ISR remains a problem, especially for high risk patients, such as those with diabetes. This proposal will investigate gene delivery stents (GDS) to address these unmet needs. The therapeutic strategy for GDS in this project is based on prevention of oxidation of apolipoprotein A1 (apoA1). The program will investigate an apo-A1 gene construct that has 4 tryptophans substituted with phenylalanine (4WF), that both resists oxidation and enables apoA1 functionality for reverse cholesterol transport. A 4WF apo-A1 AAV2 gene GDS will be studied in a well characterized, severe model of diabetic atherosclerotic disease by using hypercholesterolemic diabetic swine (HDS). The HDS model requires 20 weeks to develop, prior to stent angioplasty, resulting in pigs with advanced atherosclerosis and diabetes; thus a five year project period is requested for the proposed study design. The central hypothesis of this proposal is that GDS with AAV2 encoding 4WF apoA1 will both inhibit the pathophysiology of ISR and mitigate oxidative mechanisms involved with atherogenesis in HDS. Aim 1: To formulate and characterize AAV stent-delivery components utilizing Type 2 AAV (GFP, wild type apoA1, and 4WF apoA1), and study GDS local delivery mechanisms in HDS coronary arteries. Subaim 1a. Construct vectors, and formulate AAV linker reagents. AAV2 with a CMV promoter encoding either green fluorescent protein (GFP) or wild-type human apoA1 have already been constructed, scaled up and used in our feasibility studies in healthy pigs; 4WF apo-A1 gene constructs are underway. The AAV linking system to be used will treat the stent surfaces with polyallylamine-bisphosphonate that has conjugation sites to attach Protein G with thiol reactions, followed by affinity binding of anti-AAV2 antibody for vector attachment. Subaim 1b. One week AAV2 apoA1 & 4WF studies will examine in vivo the following groups: Group 1 - control, bare metal stents; group 2 - AAV2-GFP; group 3 - AAV2-apoA1 (wild type); group 4 - AAV2-4WF apoA1 (oxidation resistant). The endpoints will include apoA1 expression, inflammation, differences in both arterial wall oxidized apoA1 and oxidized amino acid formation, and vector biodistribution. Aim 2: Perform a therapeutic study evaluating the efficacy of GDS. The study design will be: Group 1, bare metal stent (controls) or Group 2, wild type AAV2-apoA1 and Group 3, AAV2-4WF apoA1, in HDS coronary arteries. Endpoints will include anti-ISR efficacy, inhibition of apoA1 oxidation, re-endothelialization, and mitigation of regional atherosclerotic pathophysiology. The expected results will validate the therapeutic use of GDS, and will also demonstrate the superiority of the oxidant resistant 4WF apoA1 isoform in preventing post-stent pathophysiology, promoting re- endothelialization, and mitigating oxidative mechanisms that impact both restenosis and atherosclerosis.